The purpose of this lab is to practice column chromatography to separate enantiomers of a substance from one another. This is an essential lab technique as enantiomers in a racemic mixture can have unforeseen effects so, separating them is essential for different utilizations of the compounds. The purity is then tested using TLC plates to see how well this technique removed the enantiomers from one another.

1.In order from the bottom to the top, list the layers that will be in your column.  You can answer by following this template:  First I will put in xxx and then yyyy and then….

First I will put in a small amount of fiber glass followed by a samll layer of sand. Next, the slurry will be added which is composed of hexane and silica gel powder. Next, more hexane is added to get excess silica off the sides and allow hexane to drip through.This is followed by another small layer of sand. Powdered ferrocene is then added on top followed by one more layer of sand. Pure hexane is then put in the column as the first solvent. Then, ether is added to create a 10% ether/hexane mixture. Later, continue to add more ether until a 50:50 mixture of hexane and ether is present.

2. Generally outline what you will do in the lab. First, you will prepare your column and then what will you do and then what comes next?

First, the column will be prepared which includes placing the cotton ball, creating the slurry, and packing the column with the slurry and hexane.

Next, the sample will be loaded which includes putting in the powdered ferrocene, adding additional hexane and letting it run until 100 mL have run, adding ether to create a 10% ether/hexane mixture, and changing the solvent to a 50% ether/hexane mixture when one colored solvent reaches the end of the column.

Finally, fractional collection and analysis will be conducted. This procedure entails collecting 2 mL fractionals and analyzing every other one by TLC with the pure substance for comparison and determining their Rf values.

3. The text states that liquid chromatography is used to purify substances that have low volatility and that gas chromatography is used to purify substances that have high volatility. What does this statement mean? Your answer must include a definition of volatility. 

The volatility of a substance refers to its tendency to vaporize at any given temperature. The higher the volatility of a substance, the substance evaporates readily while on the contrary, a low volatility means the substance tends to stay in the liquid or solid phase. Gas chromatography is used to purify substances with high volatility as they will vaporize which makes it impossible to purify by liquid chromatography.

4. What force pulls the mobile phase through the stationary phase? Your answer can be a single word.

Gravity.

5. Which is the more polar solvent system? The 100% hexane or 50% hexane/50% ether. Explain.

The 50:50 hexane to ether mixture is more polar. Hexane by itself is a non-polar molecule as it's a cyclo-alkane. The ether is slightly polar due to its C-O bonds so when in solution with hexane, it will be more polar than pure hexane.

6. If you were carrying out a chromatographic resolution of 0.5 g how much silica is recommended that you use?

It's recommended to use 15.0g of silica for every 1.0g of sample. In this case, with 0.5g of chromatographic resolution, about 7.5mg of silica gel will be used.

7. What is “channeling” with respect to column chromatography?

In the case of column chromatography, channeling refers to the solvent flowing unevenly through the column creating pathways that allow the mobile phase to go through parts of the stationary phase creating inefficient resolution of the substance.

8. For the sake of time, you will be changing solvent abruptly. What can you expect to occur and why does this occur?

When changing the solvent abruptly, it can be expected to see poor separation and a loss of resolution. This may be affected in the purity of the compound as the two enantiomers may not fully separate. This occurs because it can affect the packing and the rate of purification.

9. What is the optimal flow rate for column chromatography?

The optimal velocity for the flow rate of the column chromatography is 1-2 mL/sec.

10. What should be the elution order? Will ferrocene elute first or acetyl ferrocene? Explain.

Ferrocene will elute first because it's least polar and the non-polar solvent of hexane will be used first. The acetyl-ferrocene will elute second because the polar solvent will be introduced and the acetyl-ferrocene will run down along with it.

• Started the lab by packing the column for chromatography.

• First, a small piece of cotton was placed in the bottom of the column.

• The sand was added until it leveled out over the curvature of the column.

• 15 cm tall of dry silica gel was measured out and added into a separate beaker. 

• Hexane was added to the dry silica gel until a slurry formed which acted mostly liquid in character.

• The slurry was added to the column and the solvent was drained from the column.

• The drained solvent was added back into the beaker to re-combine with excess silica gel that didn't go in the column the first time.

• This process was repeated until there was no silica gel left in the beaker and the slurry was well packed without any channels.

• A thin layer of sand was added on top of the silica gel.

• 0.5 grams of the ferrocene mixture was weighed and added to the column.

• Another thicker layer of sand was placed on top of the compound, the column was then fully packed and ready to elute.

• 50 mL of hexane was added to the column.

• The hexane dripped out of the column into an Erlynmeyer flask and was put back into the top of the column.

• This process was repeated until the first compound band was near the bottom of the column.

• 10 samples were collected every 2 mL and every other one, only 5, were analyzed.

• Once all the samples were collected a 50:50 mL hexane/ether solution was added.

• The solvent was collected and re-added until the 2nd band was near the bottom of the column. 

• The samples were collected and every other one was analyzed.

• The two samples were analyzed by TLC to determine which sample was the ferrocene or acetyl ferrocene with a 1:10 ethyl acetate/hexane

TLC analysis

From this TLC analysis, I can conclude that the first band removed from the column was ferrocene as predicted based on its polarity. The Rf values match the standard of our ferrocene along with the color. A 2nd TLC strip was utilized to further confirm the identification of the compounds using the color of the spots and their known polarities and how they would moce along the TLC strip with a polar solvent. When the other band was collected, the acetyl ferrocene. was analyzed against the standard of pure acetyl ferrocene, it also matched in relative Rf values, its inability to move with the polar solvent across the TLC plate, and its color.

The compounds were effectively collected by column chromatography as they compared very similarly to the standards and they were identified to what was predicted above and the spots were small and sharp indicating efficent isolation of the compounds. The ferrocene would be isolated first with the non-polar elute and the acetyl ferrocene would be isolated second when the eluant became more polar.

This lab was very successful in isolating the two compounds from the original mixture. This lab taught me the importance of column chromatography and its usefulness in isolating compounds with similar formulas and polarities. It was interesting to watch the compounds come down the column in different colors and move based on the polarity of the solvent entering the column.

This lab gave me good practice in remembering and applying the polarities of substances into chemistry based on the desired outcome and practicing a wildly utilized lab technique. It was interesting having so much setup in this lab with elements such as cotton and sand. I like utilizing various substances for this experiment and seeing how well it worked in isolating compounds.

1. Both Silica Gel and Alumina are common column chromatography resins. What is the chemical makeup of these resins and how would you choose between using one over the other?

Both silica gel(SiO2) and alumina(Al2O3) are polar absorbents so they allow the more polar compound to remain in the stationary phase and move down the column last. Silica is slightly acidic so it will retain basic compounds while alumina is slightly basic so it will retain acidic compounds and is better for weak to moderately polar amine compounds.

 2. The kind of chromatographic separation that you are doing is called the normal phase. How is the “reversed phase” different?

The reverse phase is the use of a non-polar stationary phase and a polar mobile phase.

3. Briefly explain flash chromatography and how it is different from gravity chromatography.

Flash chromatography is the use of air pressure and smaller silica particles to rapidly separate a compound. It's different from gravity chromatography as it uses an outside force to speed up the process rather than relying on gravity as the main driver of column chromatography.

4. Briefly explain HPLC and how it is different from gravity chromatography.

 High-performance liquid chromatography is a method used to separate liquids based on its different interactions with the stationary phase. It's different from gravity chromatography because it deals with liquids and happens with higher pressures involved.